Factors influencing boar sperm cryosurvival

Optimal sperm cryopreservation is a prerequisite for the sustainable commercial application of frozen-thawed boar semen for AI. Three experiments were performed to identify factors influencing variability of postthaw sperm survival among 464 boar ejaculates. Sperm-rich ejaculate fractions were cryopre-served using a standard freezing-thawing procedure for 0.5-mL plastic straws and computer-controlled freezing equipment. Postthaw sperm motility (assessed with a computer-assisted semen analysis system) and viability (simultaneously probed by flow cytometry analysis after triple-fluorescent stain), evaluated 30 and 150 min postthaw, were used to estimate the success of cryopreservation. In the first experiment, 168 unselected ejaculates (1 ejaculate/boar), from boars of 6 breeds with a wide age range (8 to 48 mo), were cryopreserved over a 12-mo period to evaluate the predictive value of boar (breed and age), semen collection, transport variables (season of ejaculate collection, interval between collections, and ejaculate temperature exposure), initial semen traits, and sperm quality before freezing on sperm survival after freezing-thawing. In Exp. 2, 4 ejaculates from each of 29 boars, preselected according to their initial semen traits and sperm quality before freezing, were collected and frozen over a 6-mo period to evaluate the influence of interboar and intraboar ejaculate variability in the survival of sperm after cryopreservation. In Exp. 3, 12 ejaculates preselected as for Exp. 2, from each of 15 boars with known good sperm cryosurvival, were collected and frozen over a 12-mo period to estimate the sustainability of sperm cryosurvival between ejaculates over time. Boar and semen collection and transport variables were not predictive of sperm cryosurvival among ejaculates. Initial semen traits and sperm quality variables observed before freezing explained 23.2 and 10.9%, respectively, of the variation in postthaw sperm motility and viability. However, more that 70% of total variance observed in postthaw sperm quality variables among ejaculates was explained by boar. This indicates that boar is the most important (P < 0.001) factor explaining the variability among ejaculates in sperm cryosurvival, with most (14 of the 15 boars in Exp. 3) showing consistent (P > 0.05) sperm cryosurvival over time.     

公猪精子活力和形态对受精能力的重要性

公猪的精子活力和形态评定是人工授精站的日常基本工作。本文综述了精子活力和形态评定方法的实质性进展,以及精子形态与受精能力之间的相关性。精子形态是除活力之外一个重要的受精能力关联参数,在人工授精中需要被充分考虑。     

New aspects of boar sperm encapsulation

The study takes into account the main steps and techniques for boar semen encapsulation, to optimize the instrumental insemination interventions. The use of cheap, biocompatible polymers as alginate can assure a regular, constant release of spermatozoa in the sow reproductive system, avoiding the double/triple intervention of insemination and reducing the employ of disposable materials. The encapsulation/microencapsulation of semen can therefore be the starting point of new, innovative systems of pig reproduction management.     

Efficiency of three boar sperm enrichment techniques

The objective of the study was to investigate the efficiency of three enrichment methods to separate boar spermatozoa. Twenty-four ejaculates from 12 boars (2 ejaculates/boar) were extended (30 × 10⁶ spermatozoa/mL) in commercial Beltsville Thawing Solution. Each semen sample was processed with glass wool column (GW) and glass beads (GB) filtration and with the single-layer centrifugation (SLC) technique. Semen samples before (control; C) and after treatment were evaluated for sperm CASA motility/kinetics and concentration, viability, morphology and chromatin integrity. Data were analysed with mixed models. The concentration of total and motile spermatozoa was significantly decreased after treatment in groups GW and SLC, but not in group GB. Group GW showed increased values of WOB compared with both groups C and GB. Group GB showed greater values of rapid movement spermatozoa and lower values of slow movement spermatozoa compared with group C. In group SLC, higher values of VSL, LIN and STR were observed compared with group C. In conclusion, all techniques under examination enhanced various CASA variables. Based on our results, the GB method is a promising alternative separation technique for boar sperm and deserves further research regarding swine in vitro fertilization.     

种猪精液检测中移液器的应用

随着猪人工授精技术的普及,种猪常温精液产品已被广大养猪人接受。大部分小型猪场和散养户已不再饲养种公猪,而是直接购买制作好的种猪常温精液用于配种。种猪常温精液在生产前、生产后及使用前,都必须要经过严格的质量检查和评价,质量合格的精液才能用于发情母猪输配。其中,种猪精液稀释前检查原精的活力和原精的密度尤其重要。根据国家标准《种猪常温精液》(GB-23238-2009)的要求,原精稀释后的种猪常温精液产品精液剂量为80~100毫升,精子活力≥60%,每剂量精液中直线前进运动精子数≥25亿。其中每剂量中精子数用血球计数板计数,并获得原精的密度值。     

氢化可的松对保存过程中猪精子的影响

动物长期处于应激状态会影响其生育能力。应激对雄性动物生殖影响的研究多在体内进行,但在体外研究甚少,因此本试验在体外添加糖皮质激素研究其对猪精子各项生理指标的影响。试验首先在液态保存中分别添加不同质量浓度的氢化可的松(0.1,0.25,0.5,10.0,50.0 mg/L),检测其对猪精子活力的影响。结果显示,从0.25 mg/L应激质量浓度氢化可的松开始精子活力逐渐下降且均低于对照组(P<0.05)。之后通过蛋白质印迹法在射出的猪精子中证明存在糖皮质激素受体(glucocorticoid receptor,GR)。免疫荧光检测表明GR主要在精子顶体后区及尾部中段表达。精子保存第5天时,氢化可的松+米非司酮处理组精子活力显著高于氢化可的松单独处理组(P<0.05)。随后又检测了长期(5 d)处于应激质量浓度氢化可的松作用下,精子DNA碎裂(DFI)、线粒体膜电位(MMP)、精子凋亡水平。结果表明,精子DFI增加,线粒体功能下降,凋亡比例增加,而这些作用可以被米非司酮拮抗。因此表明氢化可的松可能是通过与其受体GR结合干扰线粒体功能而影响猪精子。     

Antioxidant mechanisms and their benefit on post-thaw boar sperm quality

While being an important component of normal cellular function, excess levels of reactive oxygen species (ROS) cause cell damage and death. The ability to protect sperm against oxidative damage is of particular importance in the artificial reproduction industry because of the increased production of ROS by the sperm cell during processing. This review discusses the formation of ROS and the use of antioxidants in protecting boar sperm against oxidative damage.     

Ultrastructure of boar sperm membranes prepared by freeze-fracturing]

The objective of the present paper was to describe the topographic orientation of intramembraneous particles (IMP) in the membranes of freshly ejaculated boar spermatozoa applying the method of freeze fracturing. Disc clusters of IMP's could be distinguished in the acrosome-covering plasma membrane (PF). The border of the head to a distance of about 0.3 micron seemed to contain no IMP's (Figs. 1, 2). In the postacrosomal region in an anterior direction from the posterior ring the IMP's were found to be arranged in palissade slant rows. Statistical measurements of 20 spermatozoa (Tab. I) indicated that the slant rows extended to the greatest distance in the lateral part of the head, up to 0.67 micron (+/- 1.45-0.39) from the posterior ring. In the middle of the head the rows extended to a distance of 0.18 micron (+/- 0.10-0.42). The transition spot between the zone of sparse IMP's and the zone of densely arranged IMP's was at a distance of 1.63 microns (+/- 1.87-0.69) in an anterior direction from the posterior ring (Figs. 3, 4). In the flagellum in the plasma membrane (PF) the first spot of an ample occurrence of IMP's is located in the first mitochondrion in the spiral. The IMP clusters follow the cicumferential orientation of mitochondria in the mitochondrial spiral. The IMP's were missing in the spaces between the spiral coils (Fig. 5). The membranes of the mitochondria contain a large amount of IMP's. This activity is transferred to the clinging part of the plasma membrane (Fig. 6). A larger accumulation of IMP's can be seen at the spot where the plasma membrane covers the annulus (Fig. 7). In the plasma membrane of the main segment of the flagellum the IMP's are distributed irregularly in PF, and also in EF (Fig. 8). The EF surfaces of the plasma membrane had in general an indistinct structural organisation of IMP's. The distribution of IMP's in acrosomal membranes was found to be irregular. The postacrosomal lamina in the freeze fracturing did not look like a morphologically identifiable structure. In the nuclear envelope delimiting the posterior nuclear space, nuclear pores could be identified (Fig. 9). No nuclear pores could be seen in the nuclear envelope clinging to the posterior part of the nucleus. At this spot smaller IMP's were observed which could be a part of the proteins of the filaments connecting the concave surface of the head with the convex articular surface of the flagellum. These filaments connect the head and the flagellum (Fig. 10).(ABSTRACT TRUNCATED AT 400 WORDS)     

The acrosin system of bull, boar and ram sperm]

Described in this paper is a technique by which to separate the components of the sperma acrosin system. Included in the method are extraction of all components by means of acetic acid, separation of acrosin inhibitors on Sephadex G 100 as well as biochemical determination of proacrosin and acrosin. While species-related peculiarities were of minor importance, alterations were found to occur to the acrosin system in response to deep-freeze preservation of bull, boar, and ram sperma. Those alterations grew manifest primarily through decline in total acrosin activity and shifting of the proacrosin-acrosin ratio in the direction of proacrosin activation. Detachability of membrane-bound acrosin inhibitors was increased with significance, following in-vitro capacitation of bull sperma under heparin action.     

Capacitation and capacitation-like sperm surface changes induced by handling boar semen

Since it has been well recognized that reproductive technologies, such as cryopreservation and sex-sorting, have a detrimental impact on sperm quality. These procedures cause sperm membrane destabilization which resembles that of capacitation. The pathways of this complex biochemical event are slowly unravelling, including the vital role of coating and decoating factors on the sperm surface. Characterization of these factors is leading to the development of novel surface manipulation techniques to stabilize the sperm membrane during handling. The possible application of these for assisted pig reproduction is discussed.     

Dynamics of sperm DNA fragmentation in raw boar semen and fertility

The aims were to evaluate sperm DNA fragmentation (SDF) in boars through the dispersion of their chromatin in raw semen samples, quantifying the extent of SDF, and to assess dynamic aspects of sperm DNA damage after incubation to obtain the rate of sperm DNA fragmentation (rSDF) under thermal conditions similar to the uterus (37°C) over a period of up to 24 hr and to correlate the reproductive outcome of the sows with the SDF of the boars at ejaculation. The study was performed on a pig‐breeding farm in southern Uruguay. Sixty‐one ejaculates from five of the most frequently used hybrid boars were evaluated. Semen was collected weekly from each of the boars, using the gloved‐hand technique and discarding the jelly‐like fraction of the ejaculate. Fresh semen was kept in a water bath at 37°C and protected from light, and was thereafter processed with Sperm‐Sus‐Halomax^® to evaluate SDF. The smears for time 0 (T0) were made on farm, and thereafter smears were made at the laboratory at 4 hr of obtaining the semen (T4), then every 2 hr (T6, T8, T10, T12) and a final fixation at 24 hr (T24). Differences in SDF were observed among exposure times for all boars (p < .05), but not between T10 and T12 (p = .7751) nor T4 and T24 (p = .9113). In none of the T24 samples, sperm heads could be seen with chromatin dispersion halos. Furthermore, there were differences among boars when comparing sperm rSDF (p < .05). Farrowing rate was not affected by SDF at T0 (r = .38, p = .75), nor was litter size (r = .16, p = .70). With the present experimental conditions, we have not been able to show a relationship between sperm DNA fragmentation at ejaculation and reproductive performance. However, this could be a result of the low number of ejaculates and boars used.     

New technique of sex preselection for increasing female ratio in boar sperm model

In this study, we tried to optimize the porcine semen extender conditions to maximize the differences between live X chromosome-bearing (X) spermatozoa and to Y chromosome-bearing (Y) spermatozoa without a decline in the fertility rate at different pH conditions during storage. We observed the viability of X and Y boar spermatozoa in acidic (pH 6.2), original (pH 7.2), and alkaline condition (pH 8.2) for 5 days to investigate the effect of storage conditions on the X to Y spermatozoa ratio. The functional parameters of spermatozoa were also examined to evaluate sperm quality. Sperm motility was preserved at pH 7.2 and pH 6.2 for 3 days, while sperm motility at pH 8.2 decreased significantly after 2 days. Non-capacitated spermatozoa increased while capacitated spermatozoa decreased during storage. Sperm viability decreased significantly duration-dependent under all pH conditions, but there was no significant difference during storage at pH 6.2 and 7.2. The X: Y ratio of live spermatozoa in acidic condition was maximized (1.2:1) without affecting the sperm function and fertility-related protein expression after 2 days compared to original conditions. Moreover, insemination of sows using acidic extender increased the number of female pups on days 1 and 2 of preservation. These results indicate that the production of female offspring may increase when acidic BTS is used for 2 days without affecting the success rate of AI. Above all, this method is simple and economical compared to other methods.     

常规免疫注射对公猪精子活力的影响

精子活力影响是决定受胎率高低的重要条件。通过不同种类的疫苗对公猪精液常量和精子活力的不良影响作了较为全面的观测，为科学安排采精配种时间，提高配孕育提供了依据。     

Evaluation of the motility of boar sperm using computer technics

The motility of the spermatozoa of a normally fertile boar was measured by means of the Cellsoft Computer Automated Semen Analyzer. The following values were obtained for the chosen parameters: average speed of motion (V) 20.842 microns per second, average linearity (L) 4.960, average amplitude of lateral deflection of the head (LPH) 0.90 microns, frequency of average path intersection (FPD) 11.44 s. There were 46 spermatozoa with circular motion (27.71% of motile sperms). The majority of the spermatozoa (52%) moved at the rate of 10 to 20 microns.s-1, and linearity was most frequently in classes 3 to 4 (16%). The Semen Analyzer has brought progress in the evaluation of sperm motility; hence, the apparatus should be recommended for use in State Animal Enterprises and their A.I. stations.     

Rutin protects boar sperm from cryodamage via enhancing the antioxidative defense

This study was aimed to investigate whether and how Rutin protects boar sperm against cryoinjury during cryopreservation. Five concentrations of Rutin with 0.2, 0.4, 0.6, 1.0, and 2.0 mM were added to the freezing extender of boar sperm, respectively, and the effects on quality and function of boar sperm after freezing‐thawing were assessed. The results showed that the sperm motility, mitochondrial activity, plasma membrane integrity, and acrosomal integrity were significantly improved in 0.4 mM and 0.6 mM Rutin groups (p < .05). Compared with ganoderma lucidum polysaccharide (GLP) or Tanshinone IIA, Rutin exhibited higher rates of mitochondrial activity and acrosome integrity (p < .05). Mechanistically, the addition of Rutin at the concentration of 0.6, 0.8, and 1.0 mM significantly attenuated ROS accumulation and MDA production by improving antioxidant enzymatic activity, including SOD, CAT, and GSH‐Px (p < .05). Functionally, a higher penetration rate and the increased total efficiency of fertilization were observed in the 0.4, 0.6, and 1.0 mM Rutin groups than in the control group (p < .05). Moreover, the addition of Rutin (0.6 mM) significantly induced an increase in both the cleavage and blastocyst rates (p < .05). In summary, supplementation with Rutin in cryopreservation medium protects boar sperm against ROS attack by enhancing the antioxidative defense.     

The relationship between acrosome reaction and polyunsaturated fatty acid composition in boar sperm

This study investigated the relationship between acrosome reactions and fatty acid composition with respect to fertility in boar sperm. The acrosome reaction was induced more than 85% by 60 mM methyl-beta-cyclodextrin (MBCD), and plasma membrane integrity was significantly reduced dependent on the MBCD level in boar sperm (p < .05). The acrosome-reacted sperm exhibited significantly higher saturated fatty acids (SFAs) and lower polyunsaturated fatty acids (PUFAs) composition compared to the non-acrosome reaction group (p < .0001). In addition, the PUFAs, C22:5n-6 (docosapentaenoic acid [DPA]; p < .01) and C22:6n-3 (docosahexaenoic acid [DHA]; p < .0001) were significantly decreased, and cleavage and blastocyst formation of oocytes were significantly (p < .0001) decreased in acrosome-reacted sperm relative to non-acrosome-reacted sperm. Moreover, acrosome reaction was positively correlated with SFAs, whereas negatively correlated with PUFAs, of the PUFAs, the DPA (p = .0005) and DHA (p = <.0001) were negatively correlated with the acrosome reaction. Therefore, these results suggest that the PUFAs composition of sperm is closely involved in acrosome reaction in pigs.